Cytomegalovirus (CMV) is a common virus that infects a majority of the world's population; 60-100% of the adult population has experienced this infection and are carriers of this virus. CMV establishes latency and persistence after a primary infection and most infections are subclinical. In healthy carriers, the virus is found preferentially as a latent virus in myeloid lineage cells and reactivation is dependent of cellular differentiation into mature macrophages or dendritic cells, often caused by inflammation. Until the 1970th, only severe cases of congenital infections were thought to represent human disease of this virus. With the escalation of immunosuppressive individuals in the society such as AIDS patients and transplant patients, reactivation of CMV became a major clinical problem causing high morbidity and mortality among these patients. Therefore, the importance and the interest of CMV have increased over the past decades, as CMV disease is common in this group of patients and may be life threatening. The virus is reactivated by inflammation, and increasing evidence suggests a frequent presence of the virus in tissue specimens of patients with inflammatory diseases. Recently, increasing evidence also imply a frequent presence of an active CMV infection in cancers of different origin. We define a novel genetic variant of CMV in cancer, and demonstrate a new mechanism of cancer development.
CMV Infection and Cancer
Recent reports reveal the frequent presence of the genome and proteins of CMV in certain malignant tumors, such as colon cancer, malignant gloria (1-7), medulloblastoma (8), EBV-negative Hodgkin's lymphoma, cervix cancer, prostate cancer and breast cancer (for review see (9, 10)). We have confirmed the presence of an active CMV infection in 99% of malignant glioblastoma tumors, in >90% of medulloblastomas (MB) and neuroblastoma (NB), malignant melanoma, colon, breast, pancreas, prostate, skin, ovarian and cervix cancer (1, 8). Importantly, the virus infection remains latent in non-cancer tissue specimens obtained from the same patient, and in healthy control individuals (1, 3).
CMV and Oncomodulation:
Already in the 1970th, Fred Rapp's group reported a frequent presence of CMV in prostate cancer, and isolated a virus strain from tumors that was oncogenic in animal models (11, 12). In several later studies, CMV failed to transform normal human cells, so this virus was not considered to be oncogenic. Instead, the term oncomodulation has been proposed to describe the indirect influence of CMV on tumourigenesis mediated by numerous viral proteins with specific effects on host cell functions (reviewed in (13, 14). During the evolution, CMV has developed sophisticated mechanisms that affect many different cellular and immunological functions (15, 16). The virus produces about 170 proteins in an infected cell, of which approximately only 50 are essential for virus production (17). Thus, the vast majority of the viral proteins are devoted to control important host functions that will assist the virus to co-exist with its host. These proteins may through control of host functions contribute to cancer development, and by modulating the immune response, virus infected tumor cells will be protected from discovery by the immune system (15). CMV mechanisms potentially involved in cancer pathogenesis are referred to as oncomodulatory mechanisms (10, 13).
Oncomodulation is defined as the ability to promote, in an appropriate genetic environment supplied by tumour cells, an oncogenic process characterized by disruptions in intracellular signalling pathways, transcription factors and tumor suppressor proteins. CMV can block cellular differentiation, interfere with oncogene expression, induce specific chromosomal breaks, inhibit DNA repair mechanisms, control important epigenetic functions, control cellular proliferation, inhibit apoptosis, induce angiogenesis and cellular migration that all provide oncomodulatory mechanisms (10, 13). More recent data also suggest that this virus may be oncogenic; one study has shown that the expression of the CMV protein US28 in fact, by itself, leads to tumour development in a murine model through induced COX-2 expression and VEGF production (18, 19). Expression of US28 targeted to the intestinal epithelium in transgenic mice results in intestinal hyperplasia, adenomas and adenocarcinomas (20). In collaboration with Smits group, we recently demonstrated that US28 also leads to phosphorylation of STAT3 resulting in IL-6 production and a proliferative phenotype. STAT3 phosphorylation, for example in glioblastomas was correlated to survival in glioblastoma patients (6). We have also recently found that the CMV protein IE72 induces high telomerase activity through an interaction with SP-1 binding sites in the promoter (7). Induction of telomerase activity is a common phenomenon of oncogenic viruses. Interestingly, we found that only CMV infected cells in GBM tumors exhibited increased hTERT expression (7). In further support of an important role of CMV infection in cancer, we recently found that the level of CMV infection is associated with prolonged survival in glioblastoma patients (1). Furthermore, the level of CMV infection in glioblastoma tumors is a strong prognostic factor for patient survival. Patients with low grade CMV infection (defined as less than 25% virus positive cells) in the tumor at diagnosis survive more than 2.5 times as long as patients with high grade infection (21). In vitro, ganciclovir treatment inhibits tumour growth by 80-95% and animal models demonstrate inhibition of tumour growth by 40-75% using drugs targeting viral replication (8, 22).
CMV Avoids Detection by the Immune System
CMV has developed sophisticated mechanisms designed to avoid recognition by the immune system. For example, CMV inhibits the expression of HLA class I and class II molecules and antigen presentation, it controls T cell activation, inhibits NK cell activation, protects cells from cytolytic peptides that are released from activated T and NK cells (16, 23). CMV also produces its own and controls cellular production of chemokines, cytokines and growth factors (15). These are examples of strategies that make infected cells invisible to the immune system, and may explain why CMV infected tumors are not controlled by the immune system or by immmunotherapies developed against them. Hence, infected tumour cells will be invisible to the immune system (reviewed in (16, 23), at the same time as the virus is dependent on inflammation (24). Our group was first to identify cells of the myeloid lineage as the major circulating carriers of latent virus (24), and that immune activation of T cells and the consequent production of TNF-α and IFN-γ, resulting in macrophage differentiation, is a key element in the reactivation of latent CMV (24, 25). Virus infection also induces COX-2 (18, 26-28) and we recently found that the virus also induces 5-LO expression (29) to induce inflammation and enhance virus replication, which has a high relevance in tumour biology.
CMV Gene Products Confer Resistance to Chemotherapy
Apoptosis, or programmed cell death, is the final step in the mechanisms for killing mediated by NK cells and cytotoxic T cells. Attenuated sensitivity of tumour cells to drug-induced death is one of the major reasons for the failure of anti-cancer therapy. At least five different CMV proteins (i.e., IE1, IE2, UL36 and UL37, UL38) inhibit apoptosis and may enhance the survival of CMV-infected tumour cells. These proteins may also prevent the desired effects of chemotherapy. In support of this hypothesis, UL36 expression in neuroblastoma cells confers resistance against chemotherapy (30).
A Pivotal Role of CMV Dense Bodies in Cancer
Increasing data demonstrate that CMV proteins frequently are detected in cancers of different origin. >90 of glioblastomas, neuroblastomas, medulloblastomas, colon, breast and prostate cancer from pancreatic tumors, sarcomas, malignant melanomas, squamous cell carcinoma, ovarian cancer, cervix cancer demonstrate high CMV protein expression in tumour cells, but consistently non-tumour tissues surrounding the tumour are virus negative. We have also examined patient biopsies and found them all to be highly positive for CMV protein expression. While CMV protein expression is wide-spread in the tumours and CMV RNA transcripts are easily detected, but in sharp contrast it has been difficult to detect CMV DNA in the same tissue samples. This has been a controversial issue, puzzled many of the researchers in the field, and concerns have been raised if this represents an artefact.
Hence, there has been a long-felt need and an extensive search in the art to reveal the strategies of CMV infection and how this impacts the initiation or progression of e.g. cancer. This due to the fact that such revelation could unveil potential diagnostic methods which would allow earlier treatments of patients in need thereof, as well as potential new therapeutic treatments.